1. Field of the Invention
The present invention is related to a detecting method for detecting detection target substances within samples. The present invention is also related to magnet enveloping dielectric particles which are employed in the detecting method.
2. Description of the Related Art
Assay methods such as the sandwich method and the competition method are well known in the field of biological measurement. In the sandwich method, primary antibodies that specifically bind to antigens, which are detection target substances included in a sample, are immobilized onto the surface of a substrate. The sample is supplied onto the substrate, to cause the detection target substances to specifically bind to the primary antibodies. Next, secondary antibodies that specifically bind to the antigens and which have fluorescent labels attached thereto are caused to bind with the antigens, thereby forming so called sandwiches constituted by the primary antibodies, the antigens, and the secondary antibodies. Thereafter, fluorescence emitted by the fluorescent labels which are attached to the secondary antibodies is detected.
Evanescent fluorometry, in which fluorescence labels are excited by evanescent light, has been proposed as a method for detecting fluorescence emitted by fluorescent labels. In evanescent fluorometry, an excitation light beam is caused to enter a surface from the rear surface thereof and to be totally reflected at the front surface of the substrate. Fluorescent labels are excited by evanescent light that leaks onto the front surface of the substrate. Thereafter, fluorescence emitted by the fluorescent labels is detected.
Meanwhile, a method that utilizes the electric field enhancing effect of plasmon resonance in order to improve the sensitivity of evanescent fluorometry has been proposed in U.S. Pat. No. 6,194,223 and “Surface Plasmon Fluorescence Measurements of Human Chorionic Gonadotrophin: Role of Antibody Orientation in Obtaining Enhanced Sensitivity and Limit of Detection”, M. M. L. M. Vareiro et al., Analytical Chemistry, Vol. 77, No. 3, pp. 2426-2431, 2005. In this surface plasmon enhanced fluorometry method, a metal layer is provided on a substrate, and excitation light is caused to enter the interface between the substrate and the metal layer at an incident angle greater than or equal to a total reflection angle. Surface plasmon is generated at the metal layer by the excitation light, and fluorescence signals are amplified by the electric field enhancing effects of the surface plasmon, to improve the S/N ratio.
In addition, a method in which the electric field enhancing effects of an optical waveguide mode is utilized in order to enhance the electric field at the sensor portion, similarly to the surface plasmon enhanced fluorometry method, has been proposed in “High-sensitivity sensing of catechol amines using by optical waveguide mode enhanced fluorescence spectroscopy”, K. Tsuboi et al., Abstracts of the Spring 2007 Conference of the Academy of Applied Physics, No. 3, p. 1378, 28p-SA-4, 2007. In optical waveguide mode enhanced fluorescence spectroscopy, a metal layer and an optical waveguide layer constituted by a dielectric or the like are sequentially provided on a sensor portion. An optical waveguide mode is generated in the optical waveguide layer, and fluorescence signals are amplified by the electric field enhancing effects thereof.
In addition, U.S. Patent Application Publication No. 20050053974 and “Surface-plasmon field-enhanced fluorescence spectroscopy”, T. Liebermann and W. Knoll, Colloids and Surfaces A, Vol. 171, pp. 115-130, 2000, propose methods for detecting radiant light (SPCE: Surface Plasmon Coupled Emission), which is generated by surface plasmon induced at metal layers by fluorescence generated by fluorescent labels, instead of detecting fluorescence emitted by fluorescent labels and amplified by surface plasmon, as in the aforementioned fluorometry methods.
As described above, various methods for detecting detection target substances, which are labeled with fluorescent labels, have been proposed in the field of biological measurement.
Meanwhile, in cases that fluorescence is detected after forming sandwiches with primary antibodies which are immobilized onto substrates as described above, it is necessary to separate the sandwich combinations and secondary antibodies that have not undergone binding reactions with the detection target substance. Therefore, cleansing operations to wash away such non reactive secondary antibodies is necessary to perform measurements. Not only are the cleansing operations troublesome, but they are also a factor in increasing the amount of time required for measurements. In addition, there are cases that a portion of the detection target substance will be discarded along with supernatant liquid during the cleansing operation. Therefore, there is a possibility that the detection sensitivity will deteriorate in cases that the detection target substance is a trace component within a sample. In addition, the reaction between the detection target substance and the primary antibodies is a reaction between a solid phase surface, on which the primary antibodies are bound, and a solution (liquid phase) that includes the detection target substance. Therefore, the reaction efficiency is poor, which is another factor that prevents expedient measurements.
In this respect, Japanese Unexamined Patent Publication No. 2005-077338 proposes a method that realizes high speed measurements, does not require a cleansing operation, is capable of quantifying detection target substances, and further solves the problem of delayed reactions between the solid phase and liquid phase. Specifically, in this method, primary antibodies are labeled with magnetic particles, secondary particles are labeled with fluorescent pigment, combinations of the primary antibodies, the detection target substance, and the secondary antibodies are formed within the liquid phase without immobilizing the primary antibodies onto a substrate. The combinations are localized by magnets, to separate them from non reactive secondary antibodies, and evanescent light is irradiated onto the localized combinations to measure fluorescent signals, without undertaking a cleansing operation.
Note that paragraph [0030] of Japanese Unexamined Patent Publication No. 2005-077338 describes that it is preferable for the particle size of the magnetic particles to be 100 nm (0.1 μm) or less, from the viewpoint of dispersion properties within liquid samples, that is, in order to prevent agglomeration of the particles with each other.
Similarly, Japanese Unexamined Patent Publication No. 5(1993)-264547 proposes a sensing method that employs magnetic particles. An example is described in which fine particles having particle sizes of 100 nm or less are employed as the magnetic particles.
However, in experiments that we have conducted, expedient localization (concentration) of combinations could not be reproduced using magnetic particles having particle sizes of 100 nm or less. That is, concentration could not be achieved within several minutes, which is a level required for practical use.
Meanwhile, Japanese Unexamined Patent Publication No. 1(1989)-272970 discloses a method that employs magnetic particles having a particles size of several tens of nanometers. In this method, individual magnetic particles and combinations of the magnetic particles and a detection target substance are separated by the difference in the responses thereof with respect to a magnet, that is, by the difference in concentration speeds, to measure signals from the combinations.
Detecting methods that employ localization by magnetic particles are extremely attractive as biological measurement methods, because they enable reactions in the liquid phase and they obviate cleansing operations to separate combinations of substances and non reactive secondary antibodies. However, these methods have remained merely concepts, and were not put into practical use.
As a result of investigation into the reasons why such methods are not practical, the resent inventors discovered the following problems which are encountered when actually practicing localization of combinations using magnetic particles.    1) Depending on the storage conditions of the magnetic particles, there is a possibility that the magnetic particles are magnetized and agglomerate prior to use in detection, which leads to deterioration in dispersion properties during use.    2) In the case that the magnetic particles include metal materials, there is a possibility that metal quenching, a phenomenon in which optical signals are absorbed by metals, will occur when the magnetic particles come into the vicinities of photoresponsive labels. This leads to a decrease in the amount of detected optical signals, which in turn leads to deterioration (fluctuations) in the quantitative properties of the signals.